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Title: The over-expression, purification and crystallisation of the alternative oxidase
Author: Elliott, Catherine Rebecca
Awarding Body: University of Sussex
Current Institution: University of Sussex
Date of Award: 2013
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The alternative oxidase (AOX) is an integral monotopic membrane protein which branches from respiratory chain at the point of the Q-pool in the mitochondria of all flowers, some fungi, and some protists such as the human parasite Trypanosoma brucei. The aim of this project is threefold: to establish an over-expression and purification protocol for recombinant Sauromatum guttatum alternative oxidase (SgrAOX); to use expressed SgrAOX for structural analysis such as crystallography; and finally to use in silico methods to model the alternative oxidase protein. Of these three, only the first and last have been attempted previously, with varying success. The second, namely structural analysis, has never been attempted with SgrAOX. In order to achieve the aims of this project, primarily laboratory-based protein production were used, in conjunction with downstream analysis using structural biology techniques. The in silico modelling was carried out using a wide range of algorithms freely available on the World Wide Web. Results of this project are: the determination of an over-expression system and purification protocols in two E.coli strains, producing enough protein to use for the second objective detailed above. While no crystal structure has been obtained, significant steps toward identifying a protocol for rAOX crystallisation have been made. Results from structural analysis support modelling predictions and give novel insights into the thermostability of the protein. New and detailed homology models have been created and critically evaluated, with a very recent crystal structure from our collaborators providing a unique set of data for model evaluation. The outcome of this project has contributed towards the determination of conditions under which SgrAOX protein may form crystals, and therefore bringing the acquisition of a SgrAOX protein structure closer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD0415 Biochemistry